Building a eukaryotic chromosome arm by de novo design and synthesis

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a “one-amino-acid-one-codon” strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

They then went even further to recode these genes with a very aggressive recoding scheme coupled with synthetic transcription control elements.They devised a very smart selection scheme to test and validate variable combinations of the coding sequences and control elements and successfully identified working combinations for 24 of the 25 genes, with one not tested due to failed assembly.
They then showed that the synthetic linear chromosome can function by itself, despite with significant growth defects, which is expected considering the importance of codon choices.

It is a great work!
Would recommend for publication.
However, some revision can definitely make the paper clearer to the readers.
In particular: 1.A more thorough description regarding why the left arm of yeast chromosome XII is chosen as the testing bed would be very helpful.Currently it seems very random.For whatever criteria, it would be better to show the comparison of this chosen region to the rest of yeast genome.
2. 150-151 Why the CYC1 reporter is chosen?3. 413-414 Understand it is given as a reference, but it would still be ideal to briefly discuss the design principles of the recoding scheme and how GeneDesign generates it.A detailed information of which codon is used for which amino acid would be needed here.Also, ideally explanation as why the codon is chosen out of other synonymous codons.4. A sequence list of promoters and terminators would be needed.5. 470-472 What happened to UIB4 on ptSYN24 in comparison to ptWT25?Please clarify?Is the wildtype copy of UIB4 used in ptSYN24?Or is the UIB4 simply not included in ptSYN24?
In general, again, great work!Reviewer #2: Remarks to the Author: In this manuscript, the authors report the minimization of yeast chromosome XII left arm (chrXIIL, 150 kb) by introduction of an episomal essential gene array (10 genes) and chromosomal truncation, and the impact of these operations is analyzed in details.
However, the research ideas and technical strategy of this manuscript are very similar to two previously their published papers.The authors have already done a similar study on compacting the same chromosome arm chrXIIL, although with a slight difference in the way of chromosomal deletions (Luo et al., Genome Biol., 2021).The codon compression of 10 essential genes of chrXIIL has also been published (Liang et al., iScience, 2022).It seems like this manuscript is an upgraded version of these two articles.Furthermore, the detailed assessment of biological impacts on chrXIIL deletions in this study doesn't seem to have any significant new findings.Therefore, both the scientific significance and novelty of this work are not clear.
Reviewer #3: Remarks to the Author: This research was conducted as one of the pilot studies within the context of the international collaboration project, Sc3.0.The team's objective was to explore genome plasticity.They did this by replacing the native left arm of chromosome XII with a linear artificial chromosome.This new chromosome was designed to carry small clusters of genes that underwent various substantial modifications.These included the identification of minimal gene sets required for cell viability and the remediation of fitness defects.Additionally, the project involved recoding of promoters, terminators, and open-reading frames using a 'one-amino-acid-one codon' strategy.Despite the high-risk nature of the project, it promises high rewards, having already produced uniquely insightful results.
On the other hand, given the intricate nature of the project, it is essential to provide a clearer interpretation of certain results, particularly for readers who may not be familiar with the preceding Sc2.0 project.1. Line 76: "To facilitate genome reduction, we recently developed an iterative SCRaMbLE-based genome compaction (SGC) strategy, which allowed us to remove about 40% of synXIIL randomly while the cells remained viable at 30°C in rich medium." -"Removing about 40% of synXIIL randomly" may appear ambiguous without prior knowledge of the previous publication.Removing 40% of a chromosome in a random manner is certainly not feasible due to the essentiality of certain genes.While SCRaMbLE is conducted randomly to introduce deletions, insertions, inversions, and other changes, it does not imply that 40% of a chromosome can be arbitrarily removed.
2. Line 168: "Interestingly, we found that the telomeric TG repeats, in both neochromosomes, significantly expanded (Extended Data Fig. 2h-i), indicating the TeSS end grew a new telomere successfully, as designed."-The phrase "as designed" can be confusing in this context as it may imply that the authors were already aware that the TeSS end would grow.1j, 1k, and 1m, and I would appreciate it if the authors could provide some speculations: (a) Why did ptWT10U exhibit a higher copy number compared to ptWT10?(b) Following normalization by His3, why did YLL031C display a significantly decreased transcription level in ptWT10U-10KO compared to ptWT10-10KO?4. Line 244: "Six regions flanking essential genes not fully deleted by SCRaMbLE in our previous study were knocked out individually (Extended Data Fig. 5a)."-I find the term "not fully deleted" to be confusing.Did the authors intend to convey that SCRaMbLE only removed certain portions of the six regions but not the entire regions? 5. Regarding Extended Data Figure 5 (Line 247), I have the following questions:

I have two inquiries regarding Figure
(a) Why were only two genes labeled in the figure?(b) What was the reason for region one needing to overlap with the ORF of YLL002W? 6. Line 251, Figure 2a: "In this method, a fragment containing a universal telomere, a marker gene and a homologous region is transformed into yeast to create a new telomere at the left end of chrXII by homologous recombination."-This design is unconventional since the homology arm is present on only one side of the marker.Is this a single cross-over mechanism?It seems that this design is feasible due to the presence of a telomere on the other side of the marker, correct? 7. Line 371: "Seven out of the eight genes were also retained in ZLY348, the strain with compacted chrXIIL but retaining wild type-like growth."-Providing additional information about the compacted chrXIIL would be beneficial and add value to the understanding of the topic.8. Line 338: "YLL013C (PUF3), the gene encoding a mitochondrial outer surface protein with over 2000 putative mRNA targets, was also included." -This sentence appears unclear.Are the authors suggesting that approximately 2000 putative mRNAs will be transported into the mitochondria for translation?9.A typo in Line 376: "adn" should be "and".10.Regarding Figure 5a, top row: After the "plasmid isolation" step, why does the strain transition from a haploid feature to a heterozygous state (WT+eCDS deletion)?11.Several techniques employed in this project may not be familiar to a general audience.To enhance readability, it would be helpful to provide a couple of clarifying sentences upon their first mention.
(a) Line 193: Isoform-sequencing -It would be beneficial to explain what information can be obtained through this technique.
(b) Line 274: Genetic interaction with a target gene (GGI) -It would be valuable to elaborate on how these interactions were initially measured.
(c) Line 310: MMS and rapamycin-induced chemical stress -Providing information about which pathways are affected by MMS and rapamycin-induced chemical stress would enhance understanding.
(d) Line 361: Principal component analysis -Since this technique may not be widely known, it would be advantageous to provide a brief description of what insights can be derived from principal component analysis.

Reviewer #1 (Remarks to the Author):
High level summary: very good work.
Firstly, the authors attempted to replace the left arm of yeast chromosome XII with a prevalidated principle of using linear artificial chromosome harboring a significantly reduced set of genes.The initial attempt of using just 10 previously defined individually "essential" genes failed to yield viable clones, which is hardly surprising as scientists now appreciate the importance and intricacy of synthetic combinatorial lethal and the incomplete human understanding of gene networks and functions.The fundamental challenge is how to resubstitute in "non-essential" genes to rescue the lethality.Following a linear trajectory, the authors then systematically complemented the 10 "essential genes" with one of 49 "non-essential" genes.None of the single re-substitution worked.
What the authors demonstrated next is a good intelligence contribution.After failure to rescue non-growth phenotype with a single re-substitution, the authors "linearly" moved on with double re-substitutions of two "non-essential" genes.But here presents a fundamental mathematical challenge and impracticality of exhausting all C492 = 1,176 combinations.This challenge is further exponentially amplified as the number of "nonessential" genes needed to be added back in increase.Hence a practical, scalable solution is needed.
The authors ingeniously hypothesized using GGI as an indication to identify a small subset of genes (gene's GGI over certain thresholds) to be added back in.And it worked!The authors not only demonstrated the feasibility of identifying 2 additional genes to reconstitute in addition to the initial 10 to sustain cell viability with just 12 genes, they went further to further improve the growth phenotype with the identification of another 13 genes to a total of 25.
They then went even further to recode these genes with a very aggressive recoding scheme coupled with synthetic transcription control elements.They devised a very smart selection scheme to test and validate variable combinations of the coding sequences and control elements and successfully identified working combinations for 24 of the 25 genes, with one not tested due to failed assembly.
They then showed that the synthetic linear chromosome can function by itself, despite with significant growth defects, which is expected considering the importance of codon choices.

It is a great work!
Would recommend for publication.
However, some revision can definitely make the paper clearer to the readers.
In particular: 1.A more thorough description regarding why the left arm of yeast chromosome XII is chosen as the testing bed would be very helpful.Currently it seems very random.For whatever criteria, it would be better to show the comparison of this chosen region to the rest of yeast genome.

Response:
We thank the reviewer for the suggestion.This work is based on our previous study on the construction of a synthetic chromosome XII or SynXII (Zhang et al., Science, 2017), and initiated at the early stage during the construction of SynXII.Therefore, we just used the materials at hand to start this work.
As suggested, we compared the left arm of chromosome XII to the rest of yeast genome as following: 1) Yeast chromosome arms vary in length, ranging from 78.6 kb to about 1 Mb.The chosen chrXIIL is about 150kb in length, the seventh shortest chromosome arm in yeast, and twice the length of the shortest arm.
2) We compared the protein length distribution of chrXIIL to that of the entire yeast genome.The length of proteins encoded by this chromosome arm showed no significant difference to that of genome-wide genes (Supplementary Fig. 1a).
3) We also compared the functional complexity of the genes on chrXIIL to the ones on other arms.The average number of directly evidenced gene ontology (GO) terms per gene on chrXIIL was slightly higher than other chromosome arms (Supplementary Fig. 1b).
Accordingly, we have revised the texts as follows:  The left arm of chrXII in S. cerevisiae (chrXIIL) is 150,827 bp long, ranking as the seventh shortest yeast arm.It contains 74 genes (62 protein coding genes, 9 dubious genes, 2 pseudogenes, 1 tRNA coding gene), 3 autonomously replicating sequences and 1 Ty1 LTR (Saccharomyces Genome Database, https://www.yeastgenome.org/).Among these genes, 10 are defined as essential based on the phenotype of individual knockout mutations and their ability to support spore viability in a heterozygous diploid.The length of proteins encoded by genes on chrXIIL, showed a similar distribution pattern to that of genome-wide genes (Supplementary Fig. 1a).The average number of directly evidenced gene ontology (GO) terms per gene on chrXIIL was slightly higher than other chromosome arms, suggesting higher functional complexity to be considered during reconstruction (Supplementary Fig. 1b).

150-151 Why the CYC1 reporter is chosen?
Response: YLL035W and YLL036C share a bi-directional promoter.However, according to the previous study (Ref.21 in texts), the shared 323-bp intergenic region is not able to sustain the function of YLL036C and the coding sequence of YLL035W contains essential components of YLL036W's promoter.In the same study (Ref.21 in texts), the CYC1 promoter and terminator were used for the expression of synthetic versions of YLL035W and YLL036C, and the resulted strains did not show obvious growth defects, suggesting the CYC1 promoter could drive the expression of the two genes to support cell viability.To keep both genes functional and under similar regulation, we used the CYC1 promoter to drive the expression of YLL035W and YLL036C.
To make it clear, we have revised the texts as follows:  In addition, since YLL035W and YLL036C share a bi-directional promoter and the intergenic region is unable to sustain the function of YLL036C 21 , we chose the reported CYC1 promoter to achieve functional expression of both genes and maintain consistent regulation in ptWT10U.
3. 413-414 Understand it is given as a reference, but it would still be ideal to briefly discuss the design principles of the recoding scheme and how GeneDesign generates it.A detailed information of which codon is used for which amino acid would be needed here.Also, ideally explanation as why the codon is chosen out of other synonymous codons.
Response: Thanks for the suggestions.The information regarding which codon is utilized for each amino acid has been incorporated in Supplementary Fig. 8a.
We have revised the texts as follows:  For coding sequences (CDS), the optimized DNA sequences were generated using GeneDesign software as before 21,39 , which employs a radical codon compression scheme, in which only one optimal codon is used for each amino acid (Supplementary Fig. 8a).And the optimal codon for each amino acid was defined by the highest relative synonymous codon usage (RSCU) value in highly expressed genes in yeast genome 21,39 .4. A sequence list of promoters and terminators would be needed.
Response: Thanks for the suggestions.We have added the sequence list for 44 short synthetic promoters and 28 short synthetic terminators in Supplementary Table 3.

470-472
What happened to UIB4 on ptSYN24 in comparison to ptWT25?Please clarify?Is the wildtype copy of UIB4 used in ptSYN24?Or is the UIB4 simply not included in ptSYN24?
Response: We failed to assemble the recoded TUs of UBI4, potentially due to its repetitive nature since it encodes 5 head-to-tail ubiquitin repeats within CDS.Therefore, the UIB4 was not included in ptSYN24.
We have modified the texts as follows:  Next, based on what we learned above using native genes, we constructed the ptSYN24 neochromosome (Fig. 6a), which contains 24 recoded TUs from ptWT25 without UBI4.

Reviewer #2 (Remarks to the Author):
In this manuscript, the authors report the minimization of yeast chromosome XII left arm (chrXIIL, 150 kb) by introduction of an episomal essential gene array (10 genes) and chromosomal truncation, and the impact of these operations is analyzed in details.
However, the research ideas and technical strategy of this manuscript are very similar to two previously their published papers.The authors have already done a similar study on compacting the same chromosome arm chrXIIL, although with a slight difference in the way of chromosomal deletions (Luo et al., Genome Biol., 2021).The codon compression of 10 essential genes of chrXIIL has also been published (Liang et al., iScience, 2022).It seems like this manuscript is an upgraded version of these two articles.Furthermore, the detailed assessment of biological impacts on chrXIIL deletions in this study doesn't seem to have any significant new findings.Therefore, both the scientific significance and novelty of this work are not clear.

Response:
We appreciate the reviewer's feedback.We highlight the scientific significance and novelty of this work as follows:  Firstly, there are notable differences between the research ideas and technical strategy to simplify the chrXIIL in this study from that of our previous report in Genome Biology (Luo et al., Genome Biol., 2021).In the previous study, the SCRaMbLE-based genome compaction (SGC) method was developed to enable random deletion of chromosome regions.We solely relied on analyzing the sequences in survival strains to determine which regions in the chromosome are dispensable.However, due to the limited number of strains to be sequenced, it is not easy, if not impossible, to test the essentiality of all chromosome regions.Therefore, SGC gave us a way to quickly slim down the genome.In this study, we took a step further and tried to identify the minimal sequences/genes to substitute the entire arm.Therefore, we systematically examined the essentiality of sequences in the left arm of chrXII using CRISPR/Cas9 technology and a chromosome truncation method.Furthermore, to obtain the minimal sequences/genes, we not only tried to delete the native sequences, but also constructed an essential set of sequences/genes from scratch using cell viability as a gauge.This study led us to uncover a new fact, i.e., that only twelve genes among over 70 genes in the left arm of chromosome XII are required for cell viability.This finding has also prompted us to speculate that a similar situation may exist in other chromosomes, suggesting that the redundancy of the yeast genome is unparalleled. Secondly, as the reviewer pointed out that the codon compression of 10 individual essential genes of chrXIIL has been published in the iScience paper.However, the purpose of this study for using the recoded CDS is to test whether a native chromosome arm could be replaced totally using artificial sequences (the sequences do not exist in the nature).Therefore, we not only recoded the CDS, but also employed synthetic transcription control elements, and developed an efficient selection scheme to identify functional combinations for these genes.Our results indicate that it is possible to redesign a neochromosome using totally artificial sequences to substitute the native chromosome arm, which has never been achieved before. Thirdly, when picking genes required for cell viability, it is obvious to include the essential genes.However, besides these essential genes, there is no guidance on how to choose the other "less important" genes.In this study, we demonstrated that GGI could be used as a good indication.By including different sets of genes based on GGI, we found that 10 essential genes plus only two non-essential genes form currently the minimal gene set sufficient to replace chrXIIL for cell viability.Subsequently, we identified 13 more genes for robust fitness without numerous workloads.
Together, we hope these facts could convince the reviewer that this manuscript not just an upgraded version of the previous studies.
Reviewer #3 (Remarks to the Author): This research was conducted as one of the pilot studies within the context of the international collaboration project, Sc3.0.The team's objective was to explore genome plasticity.They did this by replacing the native left arm of chromosome XII with a linear artificial chromosome.This new chromosome was designed to carry small clusters of genes that underwent various substantial modifications.These included the identification of minimal gene sets required for cell viability and the remediation of fitness defects.Additionally, the project involved recoding of promoters, terminators, and open-reading frames using a 'one-amino-acid-one codon' strategy.Despite the high-risk nature of the project, it promises high rewards, having already produced uniquely insightful results.
On the other hand, given the intricate nature of the project, it is essential to provide a clearer interpretation of certain results, particularly for readers who may not be familiar with the preceding Sc2.0 project.1. Line 76: "To facilitate genome reduction, we recently developed an iterative SCRaMbLE-based genome compaction (SGC) strategy, which allowed us to remove about 40% of synXIIL randomly while the cells remained viable at 30°C in rich medium." -"Removing about 40% of synXIIL randomly" may appear ambiguous without prior knowledge of the previous publication.Removing 40% of a chromosome in a random manner is certainly not feasible due to the essentiality of certain genes.While SCRaMbLE is conducted randomly to introduce deletions, insertions, inversions, and other changes, it does not imply that 40% of a chromosome can be arbitrarily removed.
Response: Thanks for pointing this out.
We have changed the texts as follows:  To facilitate genome reduction, we recently developed an iterative SCRaMbLE-based genome compaction (SGC) strategy, which allowed us to remove about 40% of synXIIL while the cells remained viable at 30°C in rich medium 12 .2. Line 168: "Interestingly, we found that the telomeric TG repeats, in both neochromosomes, significantly expanded (Extended Data Fig. 2h-i), indicating the TeSS end grew a new telomere successfully, as designed."-The phrase "as designed" can be confusing in this context as it may imply that the authors were already aware that the TeSS end would grow.

Response:
We agree with the reviewer that the phrase "as designed" is confusing.
Therefore, we have deleted the phrase and revised the texts as follows:  Interestingly, we found that the telomeric TG repeats, in both neochromosomes, significantly expanded (Supplementary Fig. 2h-i), indicating the TeSS end grew a new telomere successfully 2,27 .
3. I have two inquiries regarding Figure 1j, 1k, and 1m, and I would appreciate it if the authors could provide some speculations: (a) Why did ptWT10U exhibit a higher copy number compared to ptWT10?
Response: This might be a misunderstanding of the data.We employed a two-tailed t-test to compare the copy numbers of ptWT10 and ptWT10U, and no statistically significant differences were observed (P>0.1).Therefore, our conclusion is that the copy number of the neochromosomes varies among different clones with the same genotype, and there is no significant difference between the copy numbers of ptWT10U and ptWT10.
(b) Following normalization by His3, why did YLL031C display a significantly decreased transcription level in ptWT10U-10KO compared to ptWT10-10KO?
Response: We are also curious about it.There are several possibilities: 1) The major distinction between ptWT10-10KO and ptWT10U-10KO is the alteration of the regulatory sequences of YLL035W/GRC3 and YLL036C/PRP19, which could also potentially impact the expression of YLL031C.2) Among the specific differentially expressed genes in ptWT10U-10KO, the two repressive transcription factors, YDR207C/UME6 and YDR259C/YAP6, were down-regulated (Supplementary Table 2).These two genes have been reported to be regulators of YLL031C (MacIsaac KD, et al. 2006, BMC Bioinformatics 7:113;Venters BJ, et al. 2011. Mol Cell 41(4):480-92).Another specific differentially expressed gene in ptWT10U-10KO, SUR1/YPL057C, exhibits genetic interactions with YLL035W, YLL036C, UME6 and YLL031C (SGD database).Above information suggests that the altered expression of YLL035W and YLL036C in ptWT10U-10KO may indirectly affect the expression of YLL031C.3) In comparison to BY4742, the expression of YLL031C in both ptWT10-10KO and ptWT10U-10KO was ~24.5 -fold higher, suggesting the presence of a potential feed-back mechanism to keep the expression of YLL031C below a certain threshold.Together, we speculate that the expression of YLL031C is likely influenced by multiple factors.
4. Line 244: "Six regions flanking essential genes not fully deleted by SCRaMbLE in our previous study were knocked out individually (Extended Data Fig. 5a)."-I find the term "not fully deleted" to be confusing.Did the authors intend to convey that SCRaMbLE only removed certain portions of the six regions but not the entire regions?
Response: Thanks for pointing this out.Yes, SCRaMbLE only deleted certain portions of the six regions in the strain ZLY349 by removing the gene contents between two loxPsym sites in our previous research (Luo et al., Genome Biol., 2021).ZLY349 is the viable strain with the most sequence deleted (58% of synXIIL sequences) in that study and chosen for visualization of removed parts (dashed lines, Supplementary Fig. 5a).Almost all the sequences flanking essential genes before HR II were removed in ZLY349, while the six regions following HR II were either partially deleted or retained.So, our analysis focused on examining the essentiality of these six regions.
We have modified the texts as follows:  However, further compaction attempts using the same method failed, which presumably may be partially due to the extremely slow growth of the final strain ZLY349 12 .To probe the minimal gene set to support cell viability, two additional strategies were carried out here.At first, we systematically examined the essentiality of sequences in the left arm of chrXII using CRISPR/Cas9 technology.The six regions flanking essential genes, which were either partially deleted or retained in ZLY349 12 , were knocked out individually (Supplementary Fig. 5a).Response: YLL002W and YLL006W are the two non-essential genes added to the neochromosome to maintain cell viability.They were labeled in the figure to help understanding the phenotypes of strains generated through various deletion methods.For instance, YLL002W was one of the two genes disrupted in the Δ1 strain, which showed severe growth defects.Both genes were located in region IV, between HR III and HR IV.These results also suggest the importance of these genes.

Regarding Extended Data
(b) What was the reason for region one needing to overlap with the ORF of YLL002W?
Response: In the Δ1 strain, we wanted to delete all the non-essential genes before YLL003W on the left arm of chromosome XII.To reduce the potential impact of deleting non-essential genes on neighboring essential genes, we preserved approximately 500 bp of upstream sequences and 300 bp of downstream sequences of essential genes.The intergenic region between YLL003W and YLL002W is approximately 250 bp, so that about 50bp sequences in the N terminal of YLL002W were retained.6. Line 251, Figure 2a: "In this method, a fragment containing a universal telomere, a marker gene and a homologous region is transformed into yeast to create a new telomere at the left end of chrXII by homologous recombination."-This design is unconventional since the homology arm is present on only one side of the marker.Is this a single cross-over mechanism?It seems that this design is feasible due to the presence of a telomere on the other side of the marker, correct?
Response: Yes, you are right!This is a single cross-over mechanism.And this design is feasible due to the presence of a telomere on the other side of the marker.7. Line 371: "Seven out of the eight genes were also retained in ZLY348, the strain with compacted chrXIIL but retaining wild type-like growth."-Providing additional information about the compacted chrXIIL would be beneficial and add value to the understanding of the topic.
Response: Thanks for the suggestions.
We have added more information about the compacted arm in ZLY348 in the revised texts as follows:  Seven out of the eight genes were also retained in ZLY348, the strain with a compacted chrXIIL (~40% removal of synXIIL sequences) but retaining wild type-like growth on YPD at 30°C 12 .8. Line 338: "YLL013C (PUF3), the gene encoding a mitochondrial outer surface protein with over 2000 putative mRNA targets, was also included." -This sentence appears unclear.Are the authors suggesting that approximately 2000 putative mRNAs will be transported into the mitochondria for translation?
Response: Thanks for pointing this out.Puf3p is an mRNA-bind protein that plays important roles in deadenylation-dependent mRNA decay.This protein is also involved in mitochondrion organization and localization.It is localized to the cytoplasm and the cytoplasmic side of the mitochondrial outer membrane.We are not suggesting that approximately 2000 putative mRNAs (nearly a third of the yeast transcriptome) will be transported into the mitochondria for translation.
We have modified the texts as follows:  YLL013C (PUF3), a gene that encodes an mRNA binding protein involved in mRNA decay processes and known to have over 2000 putative mRNA targets 36 , was also included (Supplementary Fig. 6f).9.A typo in Line 376: "adn" should be "and".

Figure 5 (
Line 247), I have the following questions:(a) Why were only two genes labeled in the figure?